PROJECT SUMMARY Limb-girdle muscular dystrophies (LGMDs) are a debilitating group of diseases characterized by progressive weakness of the proximal limb muscles (typically the hips and shoulders). LGMD2L, caused by recessive mutations in Anoctamin-5 (ANO5), is a common but poorly understood subtype of LGMD. The clinical course of LGMD2L often advances from difficulties in performing stressful muscle activities (e.g. heavy lifting, athletics) and generalized myalgia to loss of ambulation. The disease represents a clear and significant burden to patient quality of life. Currently, no effective treatments exist for LGMD2L; furthermore, very little is known about ANO5 function (and consequently the pathogenesis of LGMD2L), making the identification of therapeutic targets virtually impossible. Recent work has uncovered an unexpected role for other Anoctamin family members as Ca2+-activated phospholipid scramblases (Ca2+-PLSases), enzymes that facilitate movement of lipids from one leaflet of the plasma membrane bilayer to the other. This process is important for externalizing phosphatidylserine (PtdSer), which serves as a signaling molecule in a variety of physiological contexts including fusion of myoblasts to form multi-nucleated myotubes. Fusion is a central step in muscle differentiation but is impaired ANO5-/- myoblasts, which may be a key aspect of LGMD2L. While ANO5 has not been definitively characterized as a PLSase, it is highly homologous to other ANO PLSases and, as with fusion, Ca2+-dependent phospholipid scrambling (Ca2+-PLS) is reduced in cells lacking ANO5. ANO5 is thus circumstantially linked to PLS, but some evidence suggests that it may not be a resident plasma membrane protein, which would be hard to reconcile with a role as a PLSase. Furthermore, other PLSases are expressed in muscle and could be responsible for pro-fusogenic PtdSer exposure during muscle differentiation; it is likewise possible that a specific PLS-independent mechanism is used by muscle to regulate PtdSer on the outer leaflet. In order to investigate the link between ANO5, PLS, and fusion, muscle-expressed PLSases will be knocked down in a certain type of muscle precursor cell (myoblasts), which will then be induced to differentiate. Additionally, the ability of individual PLSases to overcome the fusion-deficit in ANO5-/- cells will be examined in overexpression studies (Aim 1). A proteomics-based approach will be employed to clarify role(s) of ANO5 in muscle (Aim 2). This will complement Aim 1 by elucidating ANO5 binding partners that help to mediate its relationship to PLS; more importantly, it will provide an unbiased lens through which to view ANO5 biology and LGMD2L pathology. The long-term goal of this work is to identify potential therapeutic targets for ANO5-MD by determining disease mechanisms.